UC Berkeley

Soluble guanylate cyclase (sGC) is a heme-containing heterodimeric protein which is a specific sensor of nitric oxide (NO). When stimulated by NO, sGC catalyzes the formation of 3’,5’- cyclic guanosine monophosphate (cGMP) from guanosine 5’triphosphate (GTP). The cGMP formed by sGC serves as a secondary messenger molecule which modulates numerous physiological responses including cardiovascular regulation and neurotransmission. Disruption of the NO-sGC-cGMP signaling pathway has been implicated in numerous pathologies of the cardiovascular and pulmonary systems, leading to intense research to both better understand the physiological activation of sGC and to develop small molecule therapeutics, stimulators, capable of increasing sGC activity without increasing circulating concentrations of NO.sGC is composed of two homologous α and β subunits. Each subunit contains an N-terminal heme nitric oxide oxygen binding (H-NOX) domain, Per/Arnt/Sim (PAS) and coiled-coil (CC) domains, and a C-terminal catalytic (CAT) domain. When no NO is bound at the heme (U), sGC has a low, basal level of activity. NO-binding to the heme (1-NO) increases the activity of the enzyme roughly five-fold. When concentrations of NO are in excess to the heme in sGC (xsNO), activity increases 100-fold above basal activity. Despite intense research, the mechanisms behind the excess NO activation of sGC remain unsolved. Recently solved full-length cryo-electron microscopy (cryo-EM) structures for sGC in the U and xsNO states have revealed that the CC domain plays a significant role in signal transduction in sGC. In the absence of NO, a short region of the CC domain in each subunit is bent, leading to an overall contracted conformation of the enzyme. In the presence of xsNO, the bent regions of the CC domains straighten, leading to an extension of the enzyme which is believed to contribute to the increased catalytic activity of sGC in the xsNO state. Additional cryo-EM structures soon revealed that small molecule stimulators, including FDA-approved Adempas®, bind sGC in regions which facilitate the straightening of the bent region of the CC domains. Thus, these cryo-EM structures provided the first evidence for a conformation-activity relationship in sGC.Observations of conformational change in the bent regions of the CC domains of sGC led to direct interrogation of the role of the CC domains in allosteric communication in sGC. Structure-guided mutagenesis was used to engineer two CC domain variants of sGC, one with a constitutively bent CC domain and another with a constitutively straightened CC domain. When the catalytic activities of these variant proteins were characterized, the constitutively bent protein displayed low activity even in the presence of excess NO, and the constitutively extended protein exhibited maximal activity even in the complete absence of NO. These results revealed that conformational change in the CC domains is necessary and sufficient for determining the level of sGC activity.Initial cryo-EM structures characterizing stimulator binding to sGC were performed using stimulators which shared extensive structural similarities. A structurally unique stimulator, CYR715, was found to be a potent stimulator of wild type and βC122 variants of sGC, even in the complete absence of NO. Cryo-EM structures were solved for CYR715 bound to sGC, revealing that the stimulator bound to the same sites as previously characterized stimulators, but with additional binding interactions at crucial residues in sGC which could contribute to straightening of the CC domains in NO-free conditions. Small-angle X-ray scattering data were also collected for sGC under various activating conditions, revealing that conformational extension following ligand binding in sGC is complex and cannot be directly correlated to catalytic activity.In excess NO conditions, sGC undergoes full conformational extension following a non-heme interaction with NO. Current data support a role for a reversible cysteine-NO adduct in the excess NO activation of sGC, but the exact site for this interaction has yet to be determined. Cysteine-labeling reagents were used to attempt to determine which cysteines could be responsible for this excess NO interaction, but these methods resulted in the labeling of a crucial catalytic cysteine in sGC. Bioinformatic approaches were used to investigate patterns of cysteine conservation across thousands of sGC sequences, revealing cysteines which could serve as the non-heme second site for the full activation of sGC by NO.sGC is a conformationally dynamic protein, and those conformational movements result in significant changes in catalytic activity. Conformations correlating to the basal and maximal activity states have been characterized, but the conformation(s) of the intermediate activity 1-NO state have yet to be determined. How sGC samples these conformations or how these conformations might be differentially populated during activating steps is also unknown. A model for the sGC conformational landscape, built upon structural and biochemical characterizations, is necessary to answer the remaining questions regarding sGC activation by both NO and small molecule therapeutics.